NMR analysis of a novel enzymatically active unlinked dengue NS2B-NS3 protease complex

Young Mee Kim, Shovanlal Gayen, Cong Bao Kang, Joma Joy, Qiwei Huang, Angela Shuyi Chen, John Liang Kuan Wee, Melgious Jin Yan Ang, Huichang Annie Lim, Alvin W. Hung, Rong Li, Christian G. Noble, Le Tian Lee, Andy Yip, Qing Yin Wang, Cheng San Brian Chia, Jeffrey Hill, Pei Yong Shi, Thomas H. Keller

Research output: Contribution to journalArticle

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Abstract

The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the non-structural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly 4 linker ("linked protease"), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a 1H-15N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV.

Original languageEnglish (US)
Pages (from-to)12891-12900
Number of pages10
JournalJournal of Biological Chemistry
Volume288
Issue number18
DOIs
StatePublished - May 3 2013
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Kim, Y. M., Gayen, S., Kang, C. B., Joy, J., Huang, Q., Chen, A. S., Wee, J. L. K., Ang, M. J. Y., Lim, H. A., Hung, A. W., Li, R., Noble, C. G., Lee, L. T., Yip, A., Wang, Q. Y., Chia, C. S. B., Hill, J., Shi, P. Y., & Keller, T. H. (2013). NMR analysis of a novel enzymatically active unlinked dengue NS2B-NS3 protease complex. Journal of Biological Chemistry, 288(18), 12891-12900. https://doi.org/10.1074/jbc.M112.442723